Immunological self-tolerance is critical for the prevention of autoimmunity and maintenance of immune homeostasis. The ability of the immune system to discriminate between self and non-self is controlled by mechanisms of central and peripheral tolerance. Central tolerance involves deletion of self-reactive T lymphocytes in the thymus at an early stage of development (Rocha, B. and von Boehmer, H., Science 251:1225-1228 (1991); Kisielow, P. et al., Nature 333:742-746 (1988)). Several mechanisms of peripheral tolerance have been described, including T cell anergy and ignorance (Rocha, B. and von Boehmer, H., Science 251:1225-1228 (1991); Kisielow, P. et al., Nature, 333:742-746 (1988); Schwartz, R. H., Science 248:1349-1356 (1990); Miller, J. F. A. P. and Heath, W. R., Immunol. Rev. 133:131-150 (1993)). Studies ongoing for more than a decade in rodents have provided firm evidence for the existence of a unique CD4+CD25+ population of “professional” regulatory/suppressor T cells that actively and dominantly prevent both the activation as well as the effector function of autoreactive T cells that have escaped other mechanisms of tolerance (Sakaguchi, S. et al., J. Immunol. 155:1151-1164 (1995); Takahashi, T. et al., Int. Immunol. 10:1969-1980 (1998); Itoh, M. et al., J. Immunol. 162:5317-5326 (1999)). The elimination or inactivation of these cells resulted in severe autoimmune disease, and was also found to enhance immune responses to alloantigens and even tumors (Sakaguchi, S. et al., J. Immunol. 155:1151-1164 (1995); Itoh, M. et al., J. Immunol. 162:5317-5326 (1999); Shimizu, J. et al., J. Immunol. 163:5211-5218 (1999)). Recent studies revealed that the CD4+CD25+ regulatory T cells constitute a rather homogenous population (Thornton, A. M., Shevach, E. M., J. Immunol. 164:183-190 (2000)), are derived from the thymus (Itoh, M. et al., J. Immunol. 162:5317-5326 (1999)), are naturally non-proliferative (i.e. anergic) to stimulation via the TCR, but require activation via their TCR to become suppressive and to inhibit the proliferation of CD4+ or CD8+ T cells. Once activated, however, their regulatory/suppressor function was completely antigen-nonspecific, cytokine-independent yet cell contact dependent (Thornton, A. M., Shevach, E. M., J. Immunol. 164:183-190 (2000)). The exact mechanisms of suppression, notably the cell surface and/or short-range soluble molecules involved in the T-T interaction, have yet to be characterized. New in vitro data suggest that the CD4+CD25+ T cells inhibit the proliferation of responders by inhibiting their IL-2 production (Thornton, A. M., Shevach, E. M., J. Exp. Med. 188:287-296 (1998)). Recent in vivo studies suggest that the function of CD4+CD25+ T cells is crucially dependent on signaling via the CTLA-4/CD152 molecule which was found to be constitutively expressed on CD4+CD25+ T cells (Read, S. et al., J. Exp. Med. 192:295-302 (2000); Salomon, B. et al., Immunity 12:431-440 (2000); Takahashi, T. T. et al., J. Exp. Med. 192:303-310 (2000)).
Although it has been evident for years that in rodents the CD4+CD25+ T cell population constitutes a unique lineage of “professional” regulatory/suppressor T cells which are crucial for the prevention of spontaneous autoimmune disease (Sakaguchi, S. et al., J. Immunol. 155:1151-1164 (1995)), it is unknown to date, whether CD4+4T cells exhibiting similar functional properties are naturally occurring in man. The removal and/or functional impairment of these cells in vivo in mice e.g. by anti-CD25 and/or anti-CTLA-A4 mAb treatment of animals induces various spontaneous autoimmune diseases AND rejection of tumors. The mechanism is that removal/impairment of these cells removes their negative control of autoreactive T cells so that these T cells become active. If one gives back the CD4+CD25+ T cells by adoptive transfer into these animals regulation is restored and autoimmunity/tumor rejection is stopped.
As set forth above, it is totally unknown to date whether CD4+ T cells exhibiting similar functional properties are naturally present in man. The preparation of human T cells with regulatory properties, which are, however, no CD4+CD25+ T cells is known in the art. E. g. Jonuleit, H. et al., J. Exp. Med 192:1213-1222 (2000) describe the induction of regulatory T cells from human naïve T cells by repetitive stimulation with immature dendritic cells. Most of this work was done with T cells from cord blood which is the richest source of truly naïve T cells. It is to be noted that CD4+CD25+ T cells are constitutively detectable in the human blood from early time points on. The subject of Jonuleit et al. is not a naturally occurring population. De Jong, P. et al., Int. Immunol. 6:631-638 (1994) describe the effect of TGF-β1 on naïve and memory CD4+ T cells. A differential effect is shown, with stimulatory effect on primarily activated CD45 RA+ CD4+ T cells. Proliferation of CD45 RO+ CD4+ T cells or secondary stimulated CD45 RO+ cells is suppressed. In the case of CD45 RA+ T cells TGF-β leads to an increased mean of fluorescens of CD25. The effects described here solely relate to proliferation of T cells. A regulatory capacity of naïve TGF-β treated T cells is not shown.
Surprisingly it was now found that CD4+CD25+, primarly CD45RO+ T cells (mean 6% of CD4+ T cells), hereinafter shortly referred to as “CD4+CD25+ T cells”, are present in human blood, in particular the peripheral blood of adult healthy volunteers. In the past T cells exhibiting the phenotype (i.e., suppressive/regulatory CD4+CD25+ T cells) have been known for years but they were misinterpreted to be conventional memory cells (Kanegane, H. et al., Int. Immunol., 3:1349-1356 (1991); Taka, K. et al., Immunol. 72:15-19 (1990); Jackson, A. L. et al., Clin. Immunol. Innunopathol. 54:126-133 (1990)). In contrast to previous reports the human CD4+CD25+ T cells are not conventional memory cells but rather regulatory cells exhibiting functional properties identical to their rodent counterparts. CTLA-4 (CD152), for example, which is essential for the in vivo suppressive activity of CD4+CD25+ T cells, was constitutively expressed, and remained strongly upregulated after stimulation. The cells were non-proliferative to stimulation via their TCR, but the anergic state was partially reversed by IL-2 and IL-15. Upon stimulation with allogeneic (but not syngeneic) mature dendritic cells or plate-bound anti CD3+anti-CD28 the CD4+CD25+ T cells released IL-10, and in coculture experiments suppressed the activation and proliferation of CD4+ and CD8+ T cells. Suppression proved IL-10 independent, yet contact dependent as in the mouse. The identification of regulatory CD4+CD25+ T cells has important implications for the study of tolerance in man, notably in the context of autoimmunity, transplantation, and cancer.